1. Field of the Invention
The invention generally relates to the treatment of mast cell (MC) and peripheral blood basophil (PBB)-associated diseases. In particular, the invention provides methods of using water soluble fullerene derivatives and targeted chimeric fullerenes to inhibit MC and PBB responses that cause allergic reactions, and other inflammatory diseases such as arthritis.
2. Background of the Invention
The Emerging Field of Nanomedicine
Nanomedicine is an emerging area of biomedical research that has potential for advancing medical science. This area of research entails the creation and use of materials at the level of molecules and atoms in order to investigate and treat diseases and disorders; generally less than 100 nM in size. The unique properties of nanomaterials, including single walled carbon nanotubes, fullerenes, quantum dots, and metal oxides, make them potential candidates for rational delivery and targeting of pharmaceutical, therapeutic, and agents for disease diagnosis, treatment, and prevention of a wide range of disease processes. Many of these molecules can be easily manipulated and functionalized by the addition of drugs or solubilizing groups within their cage structure or to their external walls and tips. This allows for specific homing to precise targets (cells, receptors, etc) related to clinical conditions to achieve the required response while minimizing side effects given their size.
The two most widely known forms of carbon include graphite and diamond. Fullerenes or “Buckyballs” represent the third allotrope of carbon(1). In this form, for example, 60 or 70 carbon molecules are arranged in a cage structure and are water insoluble unless derivatized with various compounds (FIG. 1). The fullerene family, and especially C60, has very appealing properties which can be exploited alone or through the addition of molecules within and on the outside of the cage structure.
Fullerenes also have the potential to deliver therapeutics given their desirable properties. Therapeutic and diagnostic agents can be encapsulated, covalently attached, or adsorbed on to different-sized fullerenes(2-5). These strategies help to solve drug solubility issues which is a major pitfall for many drug screening initiatives where high-throughput screening identifies new drug candidates that are bypassed due to insolubility. Thus, fullerenes may induce the re-evaluation of many by-passed drug candidates.
The small size of fullerenes and their ability to be manipulated with synthetic polymers and ligands make them attractive for specific targeting of cells and locations within the body after intravenous or subcutaneous injection. There are many possibilities for using these molecules for new therapeutic applications and improving the efficacy of drugs already developed. For example, although most microorganisms are killed by macrophages, many pathogenic organisms have developed means for resisting macrophage destruction following phagocytosis. In certain cases, the macrophage lysosome and/or cytoplasm is the obligate intracellular home of the microorganism; examples include Toxoplasma gondii, various species of Leishmania, Mycobacterium tuberculosis, and Listeria monocytogenes. Passive targeting of nanoparticulate vehicles with encapsulated antimicrobial agents to infected macrophages is being investigated as a logical strategy for effective microbial killing(6). In theory, the nanotubes act as a “Trojan horse” protecting the drug until it is released inside the cellular compartments. Adding macrophage-targeting moieties such as liposomes can increase the specificity and result in a highly specific drug delivery system.
Another field in which fullerenes are being investigated as providing new classes of drugs is in medical imaging (i.e. magnetic resonance imaging; MRI). Certain fullerenes can encapsulate metallic ions detected by imaging hardware and software while preventing the toxic metals from being absorbed into the reticuloendothelial system(7). Nanomaterials are also being investigated as being highly sensitive biosensors. Moieties can be added to nanomaterials that can be activated by changes in the environmental pH, magnetic fields, light, and heat(4;8). Given the plethora of capabilities and options that nanomaterials bring to the field of nanomedicine it is not surprising the effect it is having on medical research and drug delivery science(3).
The Effects of Fullerenes on Biological Systems
Given the relative infancy of the field, the studies examining the toxicity of fullerenes on human systems are still emerging. As with all new technologies the potential health risks for these and other nanoparticulate materials have been a concern largely due to the dearth of studies examining the effects these materials have on physiological systems. Toxicological studies mostly use uncharacterized, single wall carbon nanotubes, and the conclusions have been conflicting and inconclusive(9-13). Water solubility, dose, exposure time, and similar parameters all appear to influence the cytotoxicity of the fullerenes.
Certain fullerenes have exhibited cytotoxic effects on human cells; whereas, other fullerene derivatives have not. Unfunctionalized C60 appears to be cytotoxic in certain systems given its highly charged core structure. As more functional side chains, such as hydroxyl or carboxyl groups, are added to the fullerene skeleton, the level of cytotoxicity appears to diminish and water solubility increases(14;15). Various studies have demonstrated that fullerenes have no cytotoxic effects on keratinocytes and can protect blood mononuclear cells and macrophages from oxidative stress(16-17). Furthermore, non-derivatized, single wall carbon nanotubes showed a dose-dependent effect on pulmonary inflammation and fibrosis in mice(18) but had little effect on fibroblasts(15). In addition to functionalization, the level of cytotoxicity also depends on the concentration of fullerenes exposed to cells. Yamawaki et al. showed the cytotoxic effects of hydroxyl fullerenes on endothelial cells using high concentrations(19). In addition, high concentrations of fullerene-based amino acid nanoparticles were cytotoxic to epidermal keratinocytes, while low concentrations displayed no cytotoxic effects(20).
Fullerenes as Antioxidants
The term free radical refers to species that have momentarily accepted an extra electron which makes them highly reactive. The most common are referred to as reactive oxygen species (ROS). The ROS include free hydroxyl radicals (OH.), superoxide anions (O2−), singlet oxygen (O2), hydrogen peroxide (H2O2) and several others. These ROS can react with, cross link and alter the function of many macromolecules. Reaction products whose presence is indicative of ROS activity include 8-hydroxyl guanisine, O-tyrosine or dityrosine (indicative of protein oxidation), and malondialdehyde (indicative of peroxidation damage to phospholoipids). These molecules can bind to/complex with other macromolecules and affect a wide variety of biological processes including apoptosis, DNA mutation that cause cancers, inflammation and tissue degeneration.
Anti-oxidants are molecules that absorb the free radical electron. Superoxide dismutases are a family of enzymes that convert superoxide anion into hydrogen peroxide which is then converted into water by another enzyme, catalase. Other anti-oxidants include glutathione, Vitamins A, C, and E, and bioflavanoids.
The fullerene core can react with free radical species given its capacity to absorb electrons and disperse them through the twenty benzene rings distributed over its surface. In fact it is one of the most potent free radical scavengers known with the potential for being “sponges” in diseases involving ROS(21). This property makes them attractive therapeutic options in acute and chronic neurodegenerative diseases such as Parkinson's, Alzheimer's and Lou Gehrig's, which involve ROS probably due to the over-excitation of glutammic acid receptors(22;23).
The fullerenes must be chemically modified in order to be useful in aqueous systems. One way to modify the fullerenes is through the addition of hydroxyl groups (OH). To this end, fullerenes derivatized with OH species have been shown to prevent ischemia (poisoning due to lack of oxygen) which is initiated and propagated through sudden increases in ROS as tissues react to energy depletion(24;25). Derivatized fullerenes also reduce ROS-induced neuronal apoptosis and have been proposed as a potential therapeutic for neurogenitive disorders. Other derivatives of fullerenes include hexosulfobutyl and C3, the tris malonate derivative, and polyethylene glycol (PEG).
In short, carbon fullerenes possess several characteristics that make them appealing as agents to diagnose and fight disease, especially those with ROS involvement. There is currently a large-scale surge in fullerene research by industry and academia alike. However, the studies emerging on their toxicity are still uncertain and several issues still plague the fullerene field. First, it is difficult to predict if effective levels of fullerenes can be achieved in tissues that would affect the biological response. It is also hard to predict if the fullerenes will present hazardous side effects to other tissues besides target tissues. Lastly, it cannot be predicted if the fullerenes will be immunodetected when derivatized and exposed to serum molecules.
Regulation of Type I Hypersensitivity
Allergic reactions are the result of B cell-produced, specific IgE antibody to common, normally innocuous antigens. These antigens trigger a TH2 response in which naive T cells are induced to develop into TH2 cells in the presence of IL-4, which appears to be derived from a specialized subset of T cells, MC and PBB. These allergen-specific TH2 cells drive allergen-specific B cells to produce IgE. In simplistic terms, MC, PBB, NK cells, T cells and even B cells are responsible for driving the initial, allergen-inducing reaction through the production of IL-4, and other TH2-specific cytokines which result in IgE sensitization. Re-exposure to the allergen triggers an allergic response through the release of inflammatory mediators from MC and PBB. The IgE produced binds to FceRI on MC and PBB and the release of pre-allergic mediators is induced when two or more IgE molecules are crosslinked with allergen. Indeed, most allergy medications are aimed at neutralizing (anti-histamines, H1-receptor blockers) or preventing (anti-IgE; “Omalizumab”) MC and PBB FcεRI responses.
MC and PBB in Asthma
Mice without MC (compared to wild type or MC-depleted mice) fail to develop asthma-like pulmonary disease when sensitized with less-aggressive immunization protocols and challenged with aerosolized allergen(26;27). A characteristic feature of MC in asthmatic airways is their activated status. Elevated histamine, tryptase, leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) levels (MC mediators) in bronchoalveolar lavage fluids, the anti-histamine-sensitive bronchospastic response to inhaled adenosine (augmentation of degranulation by submaximally stimulated MC), and the ultrastructure of MC in bronchial biopsies showing an activated phenotype support the contention that MC are actively involved in asthmatic pathogenesis.
Activated MC produce a variety of mediators capable of promoting various aspects of asthma pathogenesis. IL-4 and IL-13 facilitate TH2 immunity and IgE production. Histamine, PGD2 and LTC4 increase vasopermeability and tissue edema. Histamine, LTC4 and chymase stimulate mucus production. Histamine and LTC4 lower the neurogenic threshold for irritant responses. LTC4, IL-5, TNFα, GM-CSF and various chemokines produce inflammation and target cells involved in tissue remodeling. For example, tryptase stimulates proliferation of fibroblasts, smooth muscle, endothelial cells and epithelial cells(28). Consequently, understanding new pathways for attenuating these cells to activating stimuli are worthwhile goals in the context of asthma and allergic diseases.
Basophils are recruited into the airways of asthmatics during the allergy season, and after an allergen challenge, such that increased numbers are found in induced sputum(29) and in endobronchial lung biopsies(30-32). Elevated numbers of PBB also are found in post mortem lung specimens from asthmatics(33). Basophils have long been known to participate in the late phase of the allergic response, but more recently have been demonstrated in mice to be critically involved in the delayed, chronic allergic inflammation reaction, lasting ˜1 week after a single intradermal allergen challenge, even in the absence of MC(34). Further, PBB are the predominant IL-4-producing cell 24 h after an allergen challenge(30).
MC and PBB in Arthritis
Mast cells are present in normal human synovium, but in rheumatoid arthritis (RA) and other inflammatory joint diseases this population can expand to constitute 5% or more of all synovial cells. Recent investigations in mouse models have demonstrated that mast cells have a critical role in the generation of inflammation within the joint and strongly suggest indicate that mast cells drive non-allergic immune responses, such as arthritis, as well as in allergy. MC-derived mediators cause edema, destroy connective tissue, induce lymphocyte chemotaxis and infiltration, and induce pathological fibrosis of RA joints(35-37). Moreover, MCs are involved in angiogenesis during RA, and their proteolytic activity results in cartilage destruction and bone remodeling. Indeed MC-stabilizing compounds are shown to have a beneficial effect in a RA disease model(38). Thus, mast cells appear to be critical cells of joint inflammation and targeting the MC may be one way to therapeutically treat inflammatory arthritis.
The Role of ROS in MC and PBB Mediator Release
The role of ROS species in MC and PBB-induced responses has not been investigated thoroughly. Most studies suggest that ROS is elevated following IgE stimulation. Work in rat basophil leukemic cells (RBL) has shown that stimulation through the high-affinity IgE receptor induces the production of ROS. Furthermore these endogenously produced oxidants have important functions in regulation of various MC responses, including degranulation, leukotriene secretion, and cytokine production(39;40). Conversely, antioxidants that quench intracellular ROS, differentially affect two effector functions of antigen-IgE-activated rodent MC; inhibiting degranulation and augmenting cytokine production(41). Several secretogogues induced intracellular increases of ROS levels in rodent MC(42-44), PBB(45), and human blood-derived MC(46). It has been demonstrated that ROS generation in human PBB that had released significantly more mediators when challenged with diesel exhaust particles compared to non-challenged cells(47). Some evidence indicates that allergic and inflammatory skin diseases like atopic dermatitis, urticaria and psoriasis are mediated by MC-initiated oxidative stress(48) while recent studies suggest anti-oxidants can reduce asthma symptoms in mice(49;50).
Prior Art
U.S. Pat. No. 5,994,410 (Chiang et al., Nov. 30, 1999) discloses the use of water-soluble fullerene derivatives for the treatment of some free radical-related medical conditions. However, the conditions do not include allergic reactions or inflammatory arthritis.
U.S. Pat. No. 6,265,443 (Choi et al., Nov. 30, 1999) describes methods of treating neuronal injury with carboxyfullerene, but does not describe the treatment of allergic reactions or inflammatory arthritis.
U.S. Pat. No. 6,331,532 (Murphy et al., Dec. 18, 2001) discloses antioxidant compounds that target mitochondria. The antioxidant moiety of the compound may be a derivatized fullerene.
US patent application 2006/0040938 (Hartnagel et al., published Feb. 23, 2006) describes substituted fullerenes and their use as antioxidants, but does not disclose their use to treat allergic reactions or inflammatory arthritis.
There is an ongoing need to provide methods of treating allergic reactions and inflammatory arthritis. The prior art has thus far failed to provide methods of treating allergic reactions or inflammatory arthritis that involve the use of fullerenes.